A novel pyrroleninone cross-link from bovine dentine.

The aim was to identify suspect collagen cross-links in dentine, eluting close to known cross-links in ion-exchange HPLC. Bovine tooth roots as source of dentine were powdered, demineralised, reduced, and acid-hydrolysed. Cross-linking amino acids were isolated from the acid hydrolysate by size exclusion, adsorption, and sequential ion exchange chromatography. In addition to dihydroxylysinonorleucine and hydroxylysylpyridinoline, an unknown cross-link was isolated (V-2). The ultraviolet, mass, and nuclear magnetic resonance spectra support the proposed structure of V-2, a trimeric amino acid with a pyrroleninone nucleus.

The effect of silicic acid on calcium phosphate precipitation.

So that a possible involvement in the mineralization of dental plaque could be investigated, the effects of silicic acid on calcium phosphate precipitation were assessed in vitro. By measuring the decrease in Ca2+ concentration (by means of ion-selective electrodes), we determined both spontaneous precipitation and seeded crystal growth from solutions that contained 1 mmol/L calcium, 7.5 mmol/L phosphate, 50 mmol/L Hepes pH 7.2, and various amounts of silicic acid. Polymerized silicic acid, but not its monomer, was found both to cause a 60% reduction in the lag period that precedes spontaneous precipitation and to enhance the growth rate of seeded hydroxyapatite crystals. Silica suspensions showed effects similar to those of polysilicic acid. In all cases, the precipitated material was found to be hydroxyapatite. Whereas seeded brushite crystals grew slowly without silicic acid, hydroxyapatite was the only mineral detected after crystal growth in the presence of silicic acid. Apparently, polysilicic acid acted as a substrate for hydroxyapatite nucleation, inducing secondary nuclei on both hydroxyapatite and brushite crystals. The finding that polysilicic acid could overcome part of the inhibitory effect of a phosphoprotein on calcium phosphate precipitation gave additional support for the idea that polysilicic acid and silica may promote the formation of dental calculus.

Silica-induced precipitation of calcium phosphate in the presence of inhibitors of hydroxyapatite formation.

The promotion and the inhibition of hydroxyapatite formation by various substances were determined by measurement of the induction time of spontaneous precipitation (ti) from supersaturated solutions. Silica was found to decrease ti in Hepes-buffered (pH 7.2) supersaturated solutions with a wide range of calcium-to-phosphate ratios and concentrations. Also, in suspensions of the oral bacteria S. mutans or C. matruchotii in 1 mmol/L calcium, 7.5 mmol/L phosphate, and 50 mmol/L Hepes (pH 7.2), silica was capable of stimulating precipitation. Macromolecules derived from these bacteria by freezing and thawing appeared to be strong inhibitors of calcium phosphate precipitation. In the presence of silica, the effects of these bacterial inhibitors could be partially overcome, which supports the idea that silica in dental plaque is a promoter of calculus formation. In contrast, inhibition of calcium phosphate precipitation by a low-molecular-weight inhibitor, pyrophosphate, could not be counteracted by silica.

A single-section model for enamel de- and remineralization studies. 1. The effects of different Ca/P ratios in remineralization solutions.

A prerequisite for the accurate measurement of differences between pre- and post-experimental mineral profiles in single sections by quantitative microradiography is a high degree of reproducibility of the analytical procedures. We have determined the reproducibility of both the production and analyses of microradiographs. Lesions were made in seven single bovine enamel sections and radiographed three times. Each microradiograph was then analyzed on three different occasions by use of an image analysis system. This resulted in only small standard deviations in the lesion parameters. The method was used to determine the sites of mineral deposition in lesions in single sections during remineralization in three different solutions, one of which contained 1.5 mmol/L Ca and 0.9 mmol/L PO4 (standard solution); the other two solutions were low in either Ca or PO4, but all three had the same degree of supersaturation. The mineral profiles and lesion parameters were determined after lesion formation and after remineralization for 4 days, 1, 2, and 3 weeks. The decrease in IML (integrated mineral loss) in the lesions remineralized in the standard solution was greatest during the first week. Lesion profiles revealed that, under non-standard conditions, mineral deposition was retarded in the deeper part of the lesion, in contrast to the even distribution of mineral deposition from the standard solution. Differences in IML changes and lesion profiles between these bovine enamel experimental groups diminished when remineralization continued for 2 and 3 weeks. Possibly, the reactivity of mineral surfaces in newly made lesions affected the diffusion of remineralizing ions from low concentration solutions to the lesion front.

Induction of calcium phosphate precipitation by titanium dioxide.

Titanium powder and various titanium dioxides were tested for their capacity to reduce the induction time for calcium phosphate precipitation from supersaturated solutions. Only after a pretreatment aimed at increasing its oxide surface layer was titanium powder found to accelerate the precipitation from solutions containing 2 mmol/L CaCl2, 2 mmol/L KH2PO4, 50 mmol/L Hepes, pH 7.2, and to induce precipitation from metastable solutions containing 1.2 mmol/L CaCl2, 1.2 mmol/L KH2PO4, 50 mmol/L Hepes, pH 7.2, at 37 degrees C. Even stronger effects were found when suspensions of the titanium dioxides anatase or rutile (10-50 micrograms/mL) were added to these solutions. TiO2 appeared to serve as a reactive substrate for secondary nucleation at a wide range of calcium-to-phosphate ratios and concentrations, even in the presence of 40 mg/mL bovine serum albumin, which completely inhibited precipitation in control incubations. These results suggest that the oxide surface layer of titanium implants may induce calcium phosphate precipitation in the metal-to-bone interface, which may play a role in the integration of such implants in bone.

Lipoteichoic acid inhibits remineralization of artificial subsurface lesions and surface-softened enamel.

Dental plaque produces not only acids by which underlying enamel is demineralized but also compounds which may inhibit repair of the lesions. The aim of this study was to determine how lipoteichoic acid, a bacterial compound that is abundant in dental plaque and inhibits calcium phosphate precipitation in vitro, affects the remineralization of incipient enamel lesions. Subsurface and surface-softened lesions were made in thin sections of bovine enamel, incubated with various amounts of lipoteichoic acid (isolated from Lactobacillus casei), and remineralized in 1.5 mmol/L CaCl2, 0.9 mmol/L KH2PO4, 130 mmol/L KCl, and 20 mmol/L Hepes, pH 7.0. Remineralization was followed during several weeks by repeated microradiography of the sections, and characterized by the changes in the integrated mineral loss of the lesions and the differential mineral profiles. The results showed that: (1) the effects of lipoteichoic acid on lesion remineralization were dose-dependent; (2) in subsurface lesions only the highest dose of lipoteichoic acid affected remineralization, which was delayed throughout the lesions; and (3) in surface-softened enamel, lipoteichoic acid did not affect the remineralization of the deeper parts, but remineralization of a surface zone of from 25 to 30 microns was increasingly inhibited with increasing doses. These effects were explained by different permeabilities of the surfaces of both types of lesions for the inhibitor: In subsurface lesions, lipoteichoic acid may have mainly clogged the porosities in the surface layer, whereas it could penetrate substantially into surface-softened enamel.

The effect of a fluoridated toothpaste on dentinal lesions in plaque-filled grooves: an intra-oral crossover study.

Recently, we introduced an intra-oral dentin demineralization model to evaluate the efficacy of a fluoridated toothpaste at plaque-retention sites with limited access to salivary components and fluoride. Usage of the fluoridated toothpaste reduced mineral loss from grooves in sound dentin by 15% compared with a non-fluoridated toothpaste (Lagerweij et al., 1997). In the current study, preformed dentinal lesions were subjected to similar toothpaste treatments. To improve the power of the model in discriminating between toothpaste groups, we changed the design from monadic to crossover to reduce the influence of variation between the participants. Two sets of specimens were placed in the partial prostheses of 29 participants who used a toothpaste containing either 1000 ppm fluoride or no fluoride during two consecutive six-week periods. Changes in mineral content were analyzed by transverse microradiography. The average mineral loss after the non-fluoride period was 1785 vol% x micron (SD 2399), and 1335 vol% x micron (SD 2039) after the fluoride period, a 25% inhibition in demineralization. Analysis of variance showed that the demineralization was significantly influenced not only by the type of treatment (p < 0.001 level), but also by the participant (p < 0.001), the width of the groove (p < 0.001), and the depth into the groove where changes in mineral content were measured (p < 0.017). Twenty-one percent of all the lesions showed remineralization. No significant difference in the extent or the occurrence of remineralization was found between the treatments.

The influence of the organic matrix on demineralization of bovine root dentin in vitro.

The effect of matrix degradation on the rate of demineralization of dentin lesions was investigated. It was hypothesized that the demineralized matrix would inhibit the demineralization of the underlying mineralized dentin. Bovine root dentin specimens were alternately demineralized and incubated with either a bacterial collagenase or buffer (control). The demineralization was carried out under various conditions: Acetic acid solutions were used to form incipient and advanced erosive lesions, and lactic acid solutions containing a bisphosphonate were used to form incipient subsurface lesions. Under all conditions, the demineralization was found to be accelerated when the matrix was degraded by collagenase. This increase was more pronounced in advanced erosive lesions than in incipient lesions. Microscopic examination of collagenase-treated specimens revealed that the matrix of erosive lesions contained several layers of differently affected matrices, whereas the matrix of subsurface lesions appeared to be equally affected throughout the lesion. In conclusion, the matrix degradation was different in erosive and subsurface lesions but promoted the demineralization in both types of lesions.

Modification of amino acid residues in carious dentin matrix.

The Maillard reaction between sugar and protein has been postulated as the cause for the browning and arrestment of caries lesions. This reaction has been implicated as the cause for decreased degradability of collagen in vivo. The aim of the present study was to verify the occurrence of the reaction in vivo. Carious and sound dentin samples were taken from extracted human teeth and analyzed for the fluorescence characteristic of the Maillard reaction and oxidation and, by HPLC, for Maillard products. In addition, physiological cross-links were analyzed by HPLC. Oxidation- and Maillard reaction-related fluorescence increased in collagenase digests from carious dentin. Advanced Maillard products (carboxymethyllysine and pentosidine) increased, whereas furosine, a marker for the initial reaction, was not observed consistently. This implies no direct addition of sugars to protein, but rather the addi-tion of smaller metabolites and glycoxidation products. In addition, the physiological cross-links hydroxylysinonorleucine and dihydroxylysinonorleucine decreased in carious dentin. Also for hydroxylysylpyridinoline, a decrease was observed, but not consistently. In conclusion, the caries process modifies amino acids in dentin collagen, which can lead to increased resistance against proteolysis and ultimately to caries arrestment.

Inhibition of hydroxyapatite crystal growth by lipoteichoic acid.

Lipoteichoic acids were purified from freshly isolated strains of Streptococcus sanguis and Lactobacillus casei, and from a laboratory strain of Strep. rattus BHT. Their effects on the seeded growth of hydroxyapatite in 1 mM CaCl2, 7.5 mM KH2PO4, 50 mM HEPES (pH 7.2) were determined. At lipoteichoic acid concentrations of 25 microM organic phosphate (Po) and higher, the growth of hydroxyapatite appeared to be inhibited, at 100 microM Po inhibition was complete for several hours. In contrast, low concentrations of lipoteichoic acids (5-10 microM Po) may have a stimulatory effect on the seeded growth of hydroxyapatite. It was concluded that lipoteichoic acid may be one of the bacterial products that inhibit enamel lesion repair in vivo.

Adsorption of [3H]-lipoteichoic acid to hydroxyapatite and its effect on crystal growth.

3H-labelled lipoteichoic acid was isolated from Lactobacillus casei cells cultured in the presence of [2-3H]-glycerol. The adsorption characteristics of both native and deacylated [3H]-lipoteichoic acid on to hydroxyapatite crystals were determined in 2.0 mM CaCl2, 50 mM Hepes, pH 7.2. The maximum numbers of adsorption sites per unit surface area were 80 +/- 3 and 49 +/- 2 nmol/m2 for native and deacylated lipoteichoic acid, respectively. At low concentrations, which were previously found to stimulate hydroxyapatite growth in a supersaturated solution (1.0 mM CaCl2, 7.5 mM KH2PO4, 50 mM Hepes, pH 7.2), lipoteichoic acid appeared for a large part adsorbed to the crystals, while crystal growth was delayed when there was an excess of unbound lipoteichoic acid in solution. These results suggest that lipoteichoic acid released from plaque bacteria may have dual effects both on the precipitation of calcium phosphate in dental plaque and on the remineralization of lesions in the underlying hard tissues.

Degradation of bovine incisor root collagen in an in vitro caries model.

The effects of pH, ionic strength and proteinases on the destruction of bovine incisor root collagen were studied. Experiments were done with powdered and intact root specimens. Completely demineralized root powder was subjected to solutions of varying pH and ionic strength: (a) 0.1 M acetic acid, pH 4.0, (b) 0.1 M acetic acid + 0.15 M KCl, pH 4.0, (c) 0.1 M Hepes, pH 7.0 or to (d) 0.1 M Hepes + 0.15 M KCl, pH 7.0 at 37 degrees C. The surfaces of intact root specimens were exposed to 0.1 M acetic acid, pH 4.0 (which resulted in erosive lesions) or to 0.1 M lactic acid, 0.2 mM methane hydroxy diphosphonate, pH 5.0 (which produced subsurface lesions) at 37 degrees C. After incubation, the extracts were analysed for soluble collagen and the insoluble matrices were treated with trypsin at 15 degrees C to determine the denatured collagen. To estimate sensitivity to non-specific proteinases, demineralized root powder was also treated with trypsin under physiological conditions of temperature, pH and ionic strength. The denaturation and subsequent solubilization of collagen material from the fibrils could be influenced by variations in pH and ionic strength but these effects were small when compared to proteolytic degradation under physiological conditions. This supports the hypothesis that, in root caries, destruction of exposed matrix collagen depends largely on the presence and activity of proteinases.

Effect of fluoride-releasing filling materials on underlying dentinal lesions in vitro.

Fluoride-releasing materials placed over carious tissue are assumed to enhance remineralisation of the underlying lesion. This remineralisation, however, also depends on the availability of calcium and phosphate, which may be supplied by the pulpal fluid. The aim of this study was to measure the fluoride release of glass ionomer cements (GICs) into underlying dentin and to measure the effect of the released fluoride on the remineralisation of the underlying dentinal lesions using transversal microradiography. Discs of fluoride-releasing GIC were placed on top of dentinal lesions in an in vitro model. The discs and the dentin slabs were covered completely by a protective layer of nail varnish, leaving only the pulpal side of the dentin slab open, and hence the dentinal tubules as the pathway for the incubation fluid to the GIC disc. Specimens were incubated in a remineralisation buffer. The materials tested were a conventional GIC, an experimental GIC that was designed to have a high fluoride release, and an inert material. Fluoride was found to penetrate through the dentin slab into the surrounding fluid. Fluoride uptake from the experimental GIC was higher than from the conventional GIC. Mineral content-depth profiles after 10 weeks' remineralisation revealed that in the outer 30 microm of the lesion a higher mineral deposition occurred for the experimental GIC than in both other groups. No differences in the overall change of integrated mineral loss were found for the tested materials. We conclude that high fluoride release from filling materials only results in superficially increased remineralisation of underlying demineralised dentin.

Effect of a fluoridated toothpaste on lesion development in plaque-filled dentine grooves: an intra-oral study.

Fluoride can inhibit caries at plaque-retention sites, but some studies indicate that fluoride is less effective in fissures than on smooth surfaces. To study the efficacy of fluoridated toothpastes at plaque-retention sites, an intra-oral model was used with bovine coronal dentine discs, in which grooves of two different widths were sawn. The discs were mounted in the partial prostheses of 31 participants divided into two groups. One group brushed with a non-fluoridated toothpaste and a second with a paste containing 1,000 ppm fluoride as NaF. After 3 months, the specimens were retrieved and from each a thin section was taken for microradiographic analysis. Lesions which developed in the grooves resembled natural lesions in terms of the presence of a surface layer and the mineral content profiles. Extensive lesions followed the direction of the dentinal tubules. The mineral loss was quantified half-way into and at the base of the grooves and ranged from 0 to 20,000 vol% x microns. Analysis of variance showed that the mineral loss was significantly influenced by the treatment and the width of the grooves (p < 0.001). In the broad grooves the average mineral loss was 19% smaller in the fluoride group than in the non-fluoride group, in the narrow grooves this value was 7%. Taking the two treatment groups together, the average mineral loss was largest half-way into the broad grooves (4,921 vol% x microns) and smallest at the base of the narrow grooves (2,289 vol% x microns). The results with this new intra-oral model indicate that the dimensions of small grooves in dentine, and thus their accessibility, determine not only their susceptibility to caries but also the protective effect of a fluoridated toothpaste.

Reproducibility of TMR for the determination of longitudinal mineral changes in dental hard tissues.

Transverse microradiography (TMR) of thin sections is the principal method for determining mineral changes in experimental lesions. Because it has become also the standard by which newly-developed methods are validated, demands on all steps of the procedure are high. Thin sections of enamel and dentin require high-precision cutting or lapping to preserve surface structures, but their different brittleness and elasticity preclude a uniform procedure. Image analysis permits the measurement of fine details at a micron scale, but because most studies are aimed at overall mineral changes, they are usually lost on broadening of the scan areas and averaging of experimental groups. Small errors in repositioning of the scan areas for repetitive measurements may yield distorted lesion profiles, while quantitative data such as the integrated mineral loss are hardly influenced. A major reason for "irreproducibility" of TMR are the different definitions of the lesion parameters as used by various research groups. We recommend that these definitions be agreed on if the full potential of TMR as an international standard is to be utilized.

Uptake and release of fluoride by saliva-coated glass ionomer cement.

The 'recharging' of aged glass ionomer cement restorations with fluoride by exposing them to a concentrated NaF solution has been suggested to improve their performance as devices with slow fluoride release. We have studied the interference of salivary coatings on glass ionomer cement with the uptake of fluoride in vitro. Freshly prepared glass ionomer discs were leached in distilled water for 14 weeks, then incubated in human saliva for 2 h, 24 h or 1 week before exposure to 0.53 M NaF (1% fluoride) for 5 min. Fluoride uptake by the cement was measured indirectly as the subsequent increase in fluoride release. A 2-hour incubation in saliva reduced the fluoride uptake by half, a 24-hour incubation by 74%. The major part (93-95%) of the fluoride that was taken up was released on the first day. A 1-week salivary coating reduced the uptake by 49%, but also caused some retardation of the fluoride release: 80% of the total release was on the first day. It was concluded that despite the considerable interference by salivary coating, the amount of fluoride that can be taken up by aged glass ionomer cement remains significant. However, the fast release will limit the usefulness of such recharging in vivo.

Acidogenicity of buccal plaque after a single rinse with amine fluoride - stannous fluoride mouthrinse solution.

Caries and gingivitis prevention may benefit from chemotherapeutic plaque control, therefore we compared in a cross-over study with 5 subjects the anti-acidogenic effects of a single use of AmF-SnF2 mouthrinse solutions (Meridol with and without 5% alcohol) with baseline and with the effects of a placebo and a chlorhexidine mouthrinse (CHX). Buccal plaque was collected 0.5, 3 and 8 h after the subjects used one of the mouthrinses, each time before and after a rinse with 10% sucrose to induce lactic acid production. Samples were analysed for acid anions by capillary electrophoresis and for protein. At 0.5 h after the use of AmF-SnF2 or CHX, the concentration of acetate in resting plaque was 70% lower than at baseline or after using the placebo. Average post-sucrose acetate and lactate concentrations in the placebo group were 30-80% higher than at baseline; up to 3 h this difference was significant. 8 h after using AmF-SnF2 or CHX, the post-sucrose acetate and lactate concentrations were still 30-50% lower than after the placebo, and up to 40% lower than at baseline. To conclude, AmF-SnF2 in both Meridol formulations and CHX were shown to have a similar potency to inhibit acid production after a single rinse.

Demineralization of dentine grooves in vitro.

Caries mainly occurs at retention sites for plaque such as pits, fissures, margins of restorations and interproximal sites. An in vitro model was developed to investigate dentine caries in narrow gaps. Forty bovine coronal dentine discs were covered with bonding agent. In 20 discs, two grooves, 200 and 340 mu m wide and 500 mu m deep, were sawn to mimic fissures. In the 20 other discs, a 1-mm-wide groove was made and, after polyester sheets were placed against both walls, filled with composite. After curing of the composite and removal of the sheets, grooves of 22 mu m remained at the borders of the restoration. All grooves were exposed to 8% methylcellulose gel 0.1 M lactic acid at pH 4.8 for 1 week. Demineralization was determined by microradiography of sections sawn out of the center of the discs. The walls of the grooves showed subsurface lesions, which decreased in size towards the base of the grooves. The average mineral losses (vol % x mu m) at the entrance of the 22-, 200- and 340-mu m-wide grooves were 1,112 (SD 370), 1,277 (293) and 1,277 (255), halfway down the groove 218 (150), 659 (244) and 797 (207) and at the base of the groove 140 (88), 285 (145) and 504 (205), respectively. Analysis of variance and Tukey's B test showed that the average mineral losses from grooves of different width and from sites at different depth were all significantly different at p<0.05 level, but not for the mineral losses from the entrance of the two wider grooves and from the two lower sites in the narrowest groove. The reduced mineral loss in the two narrower groove widths is assumed to be caused by the more limited inward diffusion of acids and outward diffusion of mineral ions through the methylcellulose gel.

The Maillard reaction in demineralized dentin in vitro.

The Maillard reaction between carbohydrate and protein has been proposed as a cause of the browning of carious lesions. The aim of the present investigation was to determine the occurrence of this reaction in bovine dentin collagen in vitro and to establish the effect of the reaction on the proteolytic degradation of bovine dentin collagen in vitro. Slices of demineralized bovine dentin were incubated with 0.2 M glucose or buffer for 10 weeks at 37 degrees C. The formation of initial (furosine) and advanced (pentosidine) products of the Maillard reaction in dentin exposed to glucose was confirmed by HPLC. After reduction with NaBH4 to prevent intermediate Maillard products from further reaction, slices were either degraded with collagenase for fluorescence measurement or incubated with trypsin or pepsin to assess enzymatic degradation. Fluorescence characteristic for the Maillard reaction increased in glucose-exposed slices. Degradation of collagen by pepsin, but not by trypsin, was greatly depressed following glucose pretreatment. This may indicate an altered sensitivity to proteolytic degradation; the Maillard reaction thus has a potential role in caries arrestment.

Enzymatic release of sequestered cows' milk fluoride for analysis by the hexamethyldisiloxane microdiffusion method.

The concentrations of diffusible and total fluoride in cows' milk samples from areas with widely different fluoride levels in drinking water were determined using a fluoride electrode. The diffusible fluoride was determined by direct hexamethyldisiloxane microdiffusion while for total fluoride, samples were subjected to either open ashing or digestion with proteolytic enzymes before microdiffusion. Magnesium nitrate was studied as a new fixative for milk during open ashing and compared with magnesium acetate. Diffusible fluoride ranged from 0.024 to 0.28 microgram ml-1 while total fluoride ranged from 0.05 to 0.31 microgram ml-1. The use of proteolytic enzymes before microdiffusion resulted in total fluoride measurement. It was concluded that all fluoride in milk is inorganic in nature with the bound fluoride being physically or chemically sequestered in the milk proteins. The proposed method is convenient for total fluoride analysis in milk.